Appendix 30

Appendix 30

Modification and further validation by different laboratories of a FMDV type O specific ELISA for detection of antibodies in all susceptible species

L. Jacobs 1) ,G. Chènard2) , T. Jelsma 1) and K. de Clercq 3).

1) Cedi-Diagnostics B.V. The Netherlands 2) ID-Lelystad, the Netherlands 3) CODA, Belgium

Summary

A quick and simple Mab based ELISA for the detection of antibodies directed against type O of FMDV was developed by ID-Lelystad. The ELISA was specially developed to be used on a semi-automatic pipetting system. Specificity and sensitivity of the ELISA was previously described by G. Chènard et al. (2001). To decrease the variation between test results when used manually and to increase the specificity some slight modifications were applied. Additionally the modified Ceditest® FMDV type O ELISA was taken in production and send to different laboratories for validation.

The modified Ceditest® FMDV type O was compared with the original test using the same sera, if available. Additionally the test was validated during a workshop organised by the FAO-EUFMD commission in Sofia, Bulgaria, by the CODA-CERVA-VAR in Belgium and by the world reference laboratory of FMDV in Pirbright, England. Also the Ceditest type O ELISA was used by the BFAV Tűbingen/Riems, Germany and by the State Veterinary Institute, Lindholm in Denmark.

Test procedure and modifications

Original test Modified test

Plates coated with antigen Add 40 μl buffer + 10μl serum Plates coated with antigen Add 90 μl buffer (+5% horse serum )

and 10 μl serum Incubate 1 hour at room temperature Incubate 1 hour at room temperature

Wash the plates

Add 50 μl conjugate Add 100μl conjugate Incubate 1 hour at room temperature Incubate 1 hour at room temperature Wash the plates Wash the plates Add 100μl TMB Add 100μl TMB Stop the reaction after 15 minutes Stop the reaction after 15 minutes

Incubation time of the modified Ceditest® FMDV type O is the same, only one extra washing step is included to increase the specificity and to decrease test variation.

Calculation and interpretation of the results of the modified Ceditest® FMDV type O %inhibition = 100-((OD monster-OD blank)/OD max-OD blank)*100) Positive >= 50% Negative < 50%

Comparison of the sensitivity and specificity after modification

Table 1: Sensitivity and specificity of the Ceditest® FMDV type O before and after modification

Species Sensitivity Before Sensitivity After modification Specificity Before Specificity After modification

Bovine 96.9 (n=548) 98.2 (n=214) 95.5(n=1544) 98.9 (n=1315)

Porcine 90.8 (n=392) 85.7 (n=105)

99 (n=610) 99.7 (n=295) Ovine 96.2 (n=474) 98.8 (n=82) 94.1 (n=414) 99.8 (n=751) Average 94.6(n=1177) 95 (n=401) 96.2(n=2782) 99.5 (n=2391) Note: The virus neutralisation test was the golden standard Sera used for validation are described in detail by G. Chėnard et al., 2001. To be short: Cattle were vaccinated/infected with: O1 Manisa, O1 Hong Kong. O1 Italy, O1 Weerseloo, O1 Algarije Pigs with:O1 Italy, O1 Taiwan, O1 BFS Sheep with:O1 Marokko

Validation performed by other laboratories

Validation done during a regional workshop on FMD organised by the FAO-EUFMD commission in Sofia, Bulgaria from 18-22 March 2002

(data provided by K. de Clercq)

During this workshop 3 ELISA’s, the Checkit-FMD-3ABC, the Ceditest® FMDV type O and the SPC-ELISA with the reagents and protocols of WRL in Pirbright were practised and discussed.

Sera used during the workshop: • Sera from cattle in Turkey (n=107) vaccinated with a trivalent vaccine ( O1 Manisa, A22 and Asia 1) • Sera from an outbreak in Bulgaria (n=27) • Sera from FAO phase 16 of which 2 originated from type O vaccinated animals • Sera from type O infected cattle from Greece (n=3) • Sera from negative cattle from Greece (n=15) • Sera from negative sheep from Greece (n=9)

Results

Of the sera from Turkey 100 scored positive in the Ceditest Four sera that were negative in the Ceditest were also negative in the SPC-ELISA (Pirbright). Two sera that were negative in the Ceditest were positive in the SPC-ELISA and one serum was not tested in the SPCELISA.

From the Bulgarian outbreak all animals scored positive. Additionally the FAO sera and the type O infected cattle from Greece scored positive. Sera from negative cattle and sheep from Greece scored negative.

Overall comment: “the Ceditest ® FMDV type O was repeatable among participants and showed to be very robust”

Validation performed by CODA-CERVA-VAR, Belgium, February 2002

(data provided by K. De Clercq)

Sera used for the validation • Sequential sera of two calves vaccinated with O1 Manisa starting from day 3 post vaccination until day 63 post vaccination • Sera from negative sheep (n=80). • Sera from negative pigs (n=32). • Sera from negative cattle (n=152) • To determine the test variation a mixture of serum of three vaccinated bovids (O1, A5 and C1) were used undiluted and in different dilutions.

Results

• Specificity in pigs and sheep was 100%. Specificity in cattle was 99.6%. • Considering the sensitivity both calves tested positive from day 3 until the end of the sampling period. • Test variation (see table 2)

Table 2: Test variation Dilution Mean % inhibition

Standard deviation Undiluted 94.9 1 1:3 83.9 2.2 1:10 60.8 3.3 1:20 46.9 2.4 1:30 36.7 2.7 1:40 30.8 2.6

Validation performed by World Reference Laboratory of FMD in Pirbright, England, April 2002

(data provided by D. Paton)

Sera used for the validation • Sequential sera from 1 sheep inoculated with FMDV type O1 Greece • Field sera from a flock of sheep suspected with FMD during the 2001 outbreak (n=80) • Sequential sera from cattle vaccinated with O1 Manisa (n=60) • Sequential sera from pigs vaccinated with O1 Manisa (n=36)

Results

Table 3: Serial bleed from 1 sheep inoculated with FMDV type O1 Greece 94

Days post infection 0 1 2 3 4 5 6 8 9 10 12 14 16 20 23 PI SPC-ELISA with O1 Manisa as antigen 0 0 0 0 0 43 58

66 76 85 90 88 88 88 88

Bold is positive Italic is inconclusive PI Ceditest ® FMDV type O 0 1 0 9 8 42

57 78 80 82 95 98 90 95 96

Sheep field sera

Of a flock of suspected sheep 80 field sera were collected and tested in the virus neutralisation test (VNT), the SPC-ELISA and in the Ceditest® FMDV type O. In the VNT a titre of >45 is considered to be positive and of <11 is considered to be negative. Titres in between are inconclusive. In the SPC-ELISA, PI >60% are considered positive, PI <50% are considered negative and in between inconclusive. In the Ceditest® FMDV type O, PI >=50% are positive and <50% are negative. Of the 80 sheep, 68 tested negative in the as well the Ceditest as in the VNT, 10 sheep tested positive in both assays. Two sheep tested inconclusive in the VNT of which one sheep tested positive and one tested negative in the Ceditest. All sheep that tested positive in the Ceditest tested also positive in the SPC-ELISA. Three sheep that tested inconclusive in the SPC-ELISA tested negative in the VNT and in the Ceditest. One sheep that tested positive in the SPC-ELISA tested negative in the Ceditest and in the VNT.

Table 4: Cattle vaccinated with O1 Manisa Animal Days post vaccination VNT titre PI Ceditest® FMDV type O 1 0 Neg 0 21 1/64 91 2 0 Neg -2 21 1/64 61 3 0 Neg 14 21 1/90 84 4 0 Neg 7 21 1/22 32 5 0 Neg 10 21 1/512 87 6 0 Neg 9 21 1/64 62 7 0 Neg 1 21 1/32 34 8 0 Neg -1 21 1/22 47 9 0 Neg 0 21 1/22 58 10 0 Neg 20 21 1/128 85 11 0 Neg 4 21 1/32 43 12 0 Neg 2 21 1/22 46 13 0 Neg 17 21 1/32 59 14 0 Neg -1 21 1/22 48 15 0 Neg -1 21 1/32 57

Bold is positive Italic is inconclusive

All sera positive in the VNT (titre>45) tested positive in the Ceditest® FMDV type O. Of the 8 sera that were inconclusive in the VNT (titres between 11-45) 3 tested positive and 5 negative in the Ceditest® FMDV type O.

Table 5: Pigs vaccinated with O1 Manisa

Pig Days post vaccination VNT titre

PI SPC-ELISA O1 Manisa as antigen 1 0 Neg 6 28 Neg 59 2 0 Neg 6 28 Neg 64 3 0 Neg 7 28 Neg 62 4 0 Neg 17 28 1/16

90

5 0 Neg 14 28 Neg 40 6 0 Neg 16 28 Neg 83 7 0 Neg 15 28 Neg 33 8 0 Neg 10 28 Neg 54 9 0 Neg 9 28 Neg 69 10 0 Neg 29 28 Neg 54 11 0 Neg 16 28 Neg 10 12 0 Neg 18 28 neg 15 Bold is positive Italic is inconclusive PI Ceditest®FMDV type O 0

50

-2

56

-1

58

-1

95

-3 43 -4

70

-1 36 -3 35 -2

78

-1

51

1 0 0 -2

In pigs the VNT seems to be very insensitive test when compared to both the SPC-ELISA and the Ceditest® FMDV type O. All sera that tested positive in the SPC-ELISA tested positive in the Ceditest® FMDV type O. Of the 3 inconclusive sera in the SPC-ELISA, 2 tested positive and 1 tested negative in the Ceditest® FMDV type O.